The major surface protease MSP (GP63) of Leishmania sp. protozoa is comprised of a conserved group of molecules that facilitate parasite survival at critical steps in its life cycle. Homologous genes have been found in other trypanosomatid protozoa, including the TbMSP genes in Trypanosoma brucei. We previously reported that there are three classes of MSPs in Leishmania chagasi that are differentially expressed in concert with the parasite's life cycle. MSP expression and parasite virulence are coordinately up regulated during parasite growth from logarithmic to stationary phase of culture in vitro. Three TbMSP classes also exist in T. brucei. Surprisingly, inactivation of one TbMSP class (TbMSPA) by RNAi in T. brucei is lethal for cultured bloodstream forms but not for procyclic organisms, and overexpression of another class (TbMSPB) leads to death of procyclic but not bloodstream forms. Thus, the different TbMSP gene products may play distinct roles in different parasite life stages. The three MSP classes of L. chagasi may also have distinct roles, although the possibility of different functions for these classes has not been explored. All mechanisms regulating MSP expression described to date operate at a post-transcriptional level. Because of the well-characterized MSP cluster in L. chagasi, and the regulated expression ofMSP RNA and protein, L. chagasi serves as an ideal system for studying mechanisms of gene expression in Leishmania. However, the ease of transfection and the availability of RNAi as a method for functional gene knockout in T. brucei make it simpler to test some aspects of MSP expression and biology in Z brucei. In this application we propose to explore the unique post-transcriptional mechanisms regulating expression of MSP and TbMSP RNAs, and the expression of MSP and TbMSP proteins in L. chagasi and Z brucei. Our specific aims are: (1) To identify sequences that regulate the steady state levels ofMSP mRNAs in L. chagasi. (2) To compare the regulation of TbMSP genes and the functions of TbMSP proteins in T. brucei with their counterparts in L. chagasi. (3) To determine the cellular locations of the MSPs of T. brucei and L. chagasi whose coding sequences predict they lack the usual GPI anchor, and to contrast the amino acid sequences that regulate the intracellular trafficking of these unusual MSPs with the MSPs that possess a GPI membrane anchor.